Considerable effort has heretofore been devoted to analytical techniques for biomedical and industrial applications. Analytical techniques are of crucial importance in these areas of application, including clinical diagnosis. For example, over the past two decades, the relationship between cholesterol, triglyceride, and atherosclerosis has been established and clarified. Initial ascertainment of an elevation of cholesterol or triglyceride leads to more detailed evaluation of lipid and lipoprotein classes useful in diagnosis and treatment. Screening procedures for quantitation of plasma cholesterol are highly important as a primary approach to an amelioration of atherosclerosis. Because of the close association of cholesterol and atherosclerosis, multiple approaches for measurement of plasma cholesterol have been examined. Currently, colorimetric methods involving isopropanol extraction and saponification are being widely used and have been automated to allow reasonable precision and accuracy at a rate of 30 samples per hour. The colorimetric methodology is expensive, relatively non-specific, subject to interference from even slight changes in humidity and bilirubin levels, and requires the use of highly corrosive agents such as concentrated sulfuric acid, acetic anhydride and acetic acid. Colorimetric methods also require at least 5-10 cc blood sampling with requisite venipuncture.
Recently two major approaches to improve methodology for cholesterol quantitation have been made utilizing gas-liquid chromatography (GLC) and enzymatic measurement. The GLC techniques are highly specific for cholesterol, and are precise, accurate, and very sensitive. No preparatory organic solvent extraction is required, but saponification is a necessary step. The GLC methods are also true micro-methods requiring as little as 10-50 microliters of plasma, well within the range of capillary sampling. The GLC methodologies still require relatively expensive machinery which may be automated, and expert technical supervision to maintain precision.
The enzymatic methods, heretofore used, have combined two enzymes, cholesterol oxidase (CO) and cholesterol ester hydrolase (CE), with colorimetric techniques. These colorimetric methods rely on enzymatic conversion of cholesterol or its esters to cholestenone and hydrogen peroxide, and then on the reaction of the peroxide with various compounds to produce measurable chromogens or fluorogens.
About 15 years ago, enzyme-coupled electrodes were reported for the polarographic analysis of substances. For example, in my U.S. Pat. No. 3,539,455 a membrane polarographic electrode system and method was described for the rapid and accurate quantitative analysis of substances which theretofore posed difficulties in analyzing directly by polarographic methods. According to the description in my mentioned patent, small molecular substances, such as glucose were measured with a membrane polarographic electrode system. By use of cellulose or another membrane which is permeable to small molecules, such as glucose, but is impermeable to proteins, the membrane keeps glucose oxidase enzyme on the side of the membrane with the anode for reaction with glucose. Therefore, for example, if a sample of blood were placed on the membrane side opposite the electrode, with an aqueous solution of the enzyme and oxygen on the electrode side of the membrane, the low molecular weight materials, such as glucose, pass from the blood samples through the membrane for enzymatic reaction adjacent the electrode. After a certain period of time a steady state is reached when the hydrogen peroxide concentration is directly proportional to the glucose concentration and the cell produces a current flow as a function of the amount of hydrogen peroxide being formed which serves as an indication of the amount of glucose present. As disclosed in my article entitled "Electrode Systems for Continuous Monitoring in Cardiovascular Surgery", N.Y. Academy of Sciences, Vol. 102, pp. 29-45 (1962), the Clark oxygen electrode can be arranged so that it is sensitive to glucose by virtue of the fact that oxygen is consumed by enzymatic reaction in proportion to glucose content. In such arrangement, the inner membrane is impermeable to glucose and the reaction is monitored by drop in oxygen. However, my previous membrane polarographic techniques for measurement of by-product H.sub.2 O.sub.2 were limited to the detection of small molecules which were capable of permeating the membrane for enzymatic reaction with an enzyme being contained on the electrode side of the membrane.